Synthesis and Biological Activity of a New Indenoisoquinoline Copper Derivative as a Topoisomerase I Inhibitor

Topoisomerases are interesting targets in cancer chemotherapy. Here, we describe the design and synthesis of a novel copper(II) indenoisoquinoline complex, WN198. The new organometallic compound exhibits a cytotoxic effect on five adenocarcinoma cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, and DU-145) with the lowest IC50 (0.37 ± 0.04 μM) for the triple-negative MDA-MB-231 breast cancer cell line. Below 5 µM, WN198 was ineffective on non-tumorigenic epithelial breast MCF-10A cells and Xenopus oocyte G2/M transition or embryonic development. Moreover, cancer cell lines showed autophagy markers including Beclin-1 accumulation and LC3-II formation. The DNA interaction of this new compound was evaluated and the dose-dependent topoisomerase I activity starting at 1 μM was confirmed using in vitro tests and has intercalation properties into DNA shown by melting curves and fluorescence measurements. Molecular modeling showed that the main interaction occurs with the aromatic ring but copper stabilizes the molecule before binding and so can putatively increase the potency as well. In this way, copper-derived indenoisoquinoline topoisomerase I inhibitor WN198 is a promising antitumorigenic agent for the development of future DNA-damaging treatments.


Introduction
Metal-based drugs have been designed and developed for their cytotoxic effects on cancer cells since the discovery of platinum's antitumor properties [1][2][3][4].As they adopt a variety of coordination geometries, essential trace metals [5,6] are used for the implementation of metal-based complexed drugs in anticancer chemotherapies.In particular, numerous developed copper complexes have highly efficient antitumor activity [7][8][9] associated with various action mechanisms ranging from chelators [10], ionophores [11], and proteasome inhibitors [12] to inhibition of topoisomerase I and/or topoisomerase II that results in severe DNA damage, cell cycle arrest, and ultimately cancer cell death [13][14][15].
Novel topoisomerase I inhibitors, named indenoisoquinolines [16][17][18][19], were identified to have greater chemical stability compared to camptothecin and its derivatives currently used as a second-line treatment against endocrine-resistant breast cancer.The indenoisoquinoline derivatives indotecan (LMP400) and indimitecan (LMP776) have completed phase I/II clinical trials (Figure 1).WN170, could be a new efficient drug to counteract cancer cells at low doses and exerts a specific cytotoxic effect at low concentration (IC50 of 0.5 µM) on three adenocarcinoma cell lines from breast, cervix, and colon but not on non-cancerous breast MCF-10A cells and chemo-resistant pulmonary cancer H69AR cells (Figure 1) [15].To our knowledge, no studies on the presence of branching on the indenoisoquinoline side chain have been reported.Our interest in the search for anticancer organometallic drugs has been focused on the synthesis of a novel triamine ligand WN191 and its corresponding tridentate Cu(II) complex WN198 (Figure 1).The DNA interaction of this new compound was evaluated and its cytotoxicity was tested on adenocarcinoma cell lines, revealing the lowest IC50 for the triple-negative MDA-MB-231 breast cancer cell line.

Synthesis and Characterization
The synthesis of the ligand WN191 and its corresponding copper complex WN198 are described in Figure 2. Indenoisoquinoline WN191 was first obtained in a four-step reaction.Condensation of the commercially available benzo [d]indeno [1,2-b]pyran-5,11dione 1 with a primary aminoalcohol was followed by tosylation of the alcohol function to lead to compound 2 in 85% yield.The substitution of the tosyl group by the diprotected triamine and the deprotection of the Boc group led to the indenoisoquinoline derivative WN191 in 88% yield.Their complexation by copper(II) perchlorate in methanol afforded the copper indenosisoquinoline WN198 in 70% yield.In previous studies, we have shown that copper complex WN197, derived from WN170, could be a new efficient drug to counteract cancer cells at low doses and exerts a specific cytotoxic effect at low concentration (IC 50 of 0.5 µM) on three adenocarcinoma cell lines from breast, cervix, and colon but not on non-cancerous breast MCF-10A cells and chemo-resistant pulmonary cancer H69AR cells (Figure 1) [15].To our knowledge, no studies on the presence of branching on the indenoisoquinoline side chain have been reported.Our interest in the search for anticancer organometallic drugs has been focused on the synthesis of a novel triamine ligand WN191 and its corresponding tridentate Cu(II) complex WN198 (Figure 1).The DNA interaction of this new compound was evaluated and its cytotoxicity was tested on adenocarcinoma cell lines, revealing the lowest IC 50 for the triple-negative MDA-MB-231 breast cancer cell line.

Synthesis and Characterization
The synthesis of the ligand WN191 and its corresponding copper complex WN198 are described in Figure 2. Indenoisoquinoline WN191 was first obtained in a four-step reaction.Condensation of the commercially available benzo [d]indeno [1,2-b]pyran-5,11-dione 1 with a primary aminoalcohol was followed by tosylation of the alcohol function to lead to compound 2 in 85% yield.The substitution of the tosyl group by the diprotected triamine and the deprotection of the Boc group led to the indenoisoquinoline derivative WN191 in 88% yield.Their complexation by copper(II) perchlorate in methanol afforded the copper indenosisoquinoline WN198 in 70% yield.The structure of WN198 was established by single-crystal X-ray analysis.The drawing is displayed in Figure 3.The Cu complex crystallizes in the triclinic space group P1̅ with two formula units per unit cell.The XRD results show that the Cu(II) complex consists of a mononuclear [Cu(II)L(H2O)(CH3OH)] 2− (where L is the indenoisoquinolinetriamine ligand) unit and two (ClO4) − .The five coordinated Cu(II) ions occupy the center of the distorted square base of the pyramid consisting of three nitrogen atoms from the indenoisoquinolinetriamine ligand and one oxygen atom of a water molecule (1.995 Å).One O atom of (CH3)OH completes the pyramidal environment with an axial bond distance of Cu-O = 2.333 (7) Å.The structure of WN198 was established by single-crystal X-ray analysis.The drawing is displayed in Figure 3.The Cu complex crystallizes in the triclinic space group P1̅ with two formula units per unit cell.The XRD results show that the Cu(II) complex consists of a mononuclear [Cu(II)L(H2O)(CH3OH)] 2− (where L is the indenoisoquinolinetriamine ligand) unit and two (ClO4) − .The five coordinated Cu(II) ions occupy the center of the distorted square base of the pyramid consisting of three nitrogen atoms from the indenoisoquinolinetriamine ligand and one oxygen atom of a water molecule (1.995 Å).One O atom of (CH3)OH completes the pyramidal environment with an axial bond distance of Cu-O = 2.333 (7) Å.

WN191 and WN198 Display Cytotoxic Activity on Five Adenocarcinoma Cell Lines at Low Doses
Cell viability was assayed on breast cancer cells (MCF-7), triple-negative breast cancer cells (MDA-MB-231), cervix cancer cells (HeLa), colorectal cancer cells (HT-29), and prostate cancer cells (DU-145), Table 1.The IC 50 values obtained for WN191 were respectively 0.58 µM, 1.12 µM, 0.80 µM, 0.53 µM, and 1.09 µM.The copper metal significantly enhances the toxicity of the indenoisoquinoline on the triple-negative breast cancer line MDA-MB-231.For WN198, IC 50 values were lower by a factor of 3.02 for MDA-MB-231 (0.37 µM), or close to a factor of 1.11 for HeLa (0.72 µM) and 1.04 for DU-145 (1.04 µM), or slightly higher by a factor 1.53 for breast cancer hormone-dependent MCF-7 (0.89 µM) and by a factor of 2 for HT-29 (1.06 µM).All IC 50 values for compounds WN191 and WN198 were below the cisplatin values ranging from 2 to 40 µM.Compared to WN170 [15], WN198's IC 50 value was lower by a factor of 2.36 for MDA-MB-231 but not for all the other cell lines.MTS viability assays were performed on the MCF-10A human non-tumorigenic epithelial cell line, most commonly used as a model for normal human breast cells (Figure 4A).The concentration of WN198 required to inhibit 50% of the MCF-10A viability was significantly higher compared to the mean obtained from the other five adenocarcinomas' IC 50 .WN198's IC 50 was smaller compared to cisplatin but higher than that of WN197, a copper complex and a topoisomerase inhibitor [15].Additionally, we tested the ability of WN198 to alter Xenopus oocyte maturation and embryonic development, two useful single and highly organized cell systems to test metals and chemical effects [20][21][22][23].Xenopus oocytes undergo a G2/M transition, after progesterone stimulation and concomitant microinjection and balneation with WN198 (0.5, 1, and 5 µM).At those doses, oocytes treated with doxorubicin, a topoisomerase II inhibitor, displayed an inhibition of their meiosis progression (Figure 4B).Xenopus embryos could survive WN198 but not doxorubicin treatment.For 1 and 5 µM of doxorubicin, the percentage of surviving embryos was lowered at segmentation (cell division), and only half of the embryos survived gastrulation (involving cell mobility to build a three-layered organized embryo).For all doxorubicin concentrations tested, the percentage of live embryos was drastically lowered at the time of neurulation and organogenesis (Figure 4C).The viability of Xenopus embryos was followed after incubation with corresponding drugs (0.5, 1, 5 µM).Stages were identified using the Nieuwkoop and Faber table [24]: segmentation, (3 h 30 min after fertilization), gastrulation (9 h after fertilization), neurulation (19 h 45 min after fertilization), tailbud (24 h after fertilization).Experiments were performed on two independent fertilizations and 10 to 40 embryos in each condition.

WN191 and WN198 Are Concentration-Dependent Topoisomerase Inhibitors
To determine the effect of WN191 and Cu(II) complex WN198 on topoisomerase activity, in vitro tests were realized.Topoisomerase I assays rely on the relaxation of supercoiled DNA by an active topoisomerase I.With topoisomerase I, supercoiled DNA shows a relaxed profile (Figure 5).With a well-known topoisomerase I inhibitor, camptothecin (CPT), the DNA relaxation was disturbed and a part of the DNA remained supercoiled.With the addition of 1 and 2 µM of WN198 in the reactional mixture, the quantity of relaxed DNA is decreased, indicating disruption of topoisomerase I activity.WN191 disrupted topoisomerase I activity at 2 µM.DMSO (solvent control) and VP-16 (etoposide, a topoisomerase II inhibitor) have no effect on DNA relaxation induced by topoisomerase I, showing their lack of inhibitory effect on topoisomerase I activity.Experiments were performed on 10 to 20 oocytes from three different females.(C) The viability of Xenopus embryos was followed after incubation with corresponding drugs (0.5, 1, 5 µM).Stages were identified using the Nieuwkoop and Faber table [24]: segmentation, (3 h 30 min after fertilization), gastrulation (9 h after fertilization), neurulation (19 h 45 min after fertilization), tailbud (24 h after fertilization).Experiments were performed on two independent fertilizations and 10 to 40 embryos in each condition.

WN191 and WN198 Are Concentration-Dependent Topoisomerase Inhibitors
To determine the effect of WN191 and Cu(II) complex WN198 on topoisomerase activity, in vitro tests were realized.Topoisomerase I assays rely on the relaxation of supercoiled DNA by an active topoisomerase I.With topoisomerase I, supercoiled DNA shows a relaxed profile (Figure 5).With a well-known topoisomerase I inhibitor, camptothecin (CPT), the DNA relaxation was disturbed and a part of the DNA remained supercoiled.With the addition of 1 and 2 µM of WN198 in the reactional mixture, the quantity of relaxed DNA is decreased, indicating disruption of topoisomerase I activity.WN191 disrupted topoisomerase I activity at 2 µM.DMSO (solvent control) and VP-16 (etoposide, a topoisomerase II inhibitor) have no effect on DNA relaxation induced by topoisomerase I, showing their lack of inhibitory effect on topoisomerase I activity.SCDNA is used in all other reactions in the presence of Top1.(C) Etoposide (VP-16, 50 µM; topoisomerase II (Top2) poison, lane 1) is the negative control of Top1 activity inhibition, and camptothecin (CPT, 10 µM; Top1 poison, lane 2) the positive control of Top1 activity inhibition.After topoisomerase reactions, DNA is run in a 1% agarose gel, stained with ethidium bromide (0.5 µg/mL), and visualized under UV light.

WN198 Intercalates in DNA
Melting curve and fluorescence measurements were performed to confirm the results shown in Table 2 and ascertain WN191 and WN198 intercalation in DNA.
The binding affinities, determined using a fluorescence-quenching assay based on DNA-binding competition between the intercalating drug ethidium bromide and the tested molecules, were used to gain insight into the DNA-binding affinity.The apparent DNA-binding constant Kapp value of the Cu(II) complex (10.791 ± 1.638 10 7 M −1 ) is higher compared to the original ligand WN191 value (8.964 ± 0.964 10 7 M −1 ).The complexation of indenoisoquinoline ligand by copper allows a stronger interaction with DNA (Table 2).The EtBr displacement is in contrast only 2% different between the two compounds.

WN198 Intercalates in DNA
Melting curve and fluorescence measurements were performed to confirm the results shown in Table 2 and ascertain WN191 and WN198 intercalation in DNA.The binding affinities, determined using a fluorescence-quenching assay based on DNA-binding competition between the intercalating drug ethidium bromide and the tested molecules, were used to gain insight into the DNA-binding affinity.The apparent DNAbinding constant Kapp value of the Cu(II) complex (10.791 ± 1.638 10 7 M −1 ) is higher compared to the original ligand WN191 value (8.964 ± 0.964 10 7 M −1 ).The complexation of indenoisoquinoline ligand by copper allows a stronger interaction with DNA (Table 2).The EtBr displacement is in contrast only 2% different between the two compounds.

Modeling of the Interaction between WN170, WN191, and WN198 and Top1
As no experimental structures of complexes were available, we designed an approach to understand how our putative ligands could bind to Top1 and could intercalate DNA.The starting point was the crystal structure of human DNA Top1 in complex with topotecan poison (Figure 6A) (PDB ID: 1K4T, 2.1 Å).Based on the coordinates of the poison, we subsequently modeled the interaction of WN170, WN191, and WN198 by molecular docking.The full procedure of molecular modeling is detailed in Section 4.7.
The starting point was the crystal structure of human DNA Top1 in complex with topotecan poison (Figure 6A) (PDB ID: 1K4T, 2.1 Å).Based on the coordinates of the poison, we subsequently modeled the interaction of WN170, WN191, and WN198 by molecular docking.The full procedure of molecular modeling is detailed in Section 4.7.
On one hand, based on these models, the previously described WN170 adopts a similar conformation to the commercial poison topotecan which has an aromatic moiety intercalating the DNA (Figure 6B).The relatively small branched chain of WN170 can easily fit the binding cavity.On the other hand, the complex between WN191 and Top1 is similar to the previous one with the aromatic moiety totally fitting the DNA groove (Figure 6C).Nevertheless, one can observe that the larger branched arm of WN191 had to switch to another position, putatively decreasing with a small impact on the affinity of this molecule.Moreover, WN198 has a similar structure as WN191 with a copper ion chelating a water molecule.This interaction was difficult to model by molecular mechanics as copper ions are not well parametrized in a classical forcefield.One model was proposed (Figure 6D) that can be easily superimposed on the ligand WN191.Although the binding modes of WN191 and WN198 are almost the same, their activities are slightly different, which may be explained by copper ion playing a role in the stabilization of the ligand before the binding to the DNA groove as discussed in Section 3.  On one hand, based on these models, the previously described WN170 adopts a similar conformation to the commercial poison topotecan which has an aromatic moiety intercalating the DNA (Figure 6B).The relatively small branched chain of WN170 can easily fit the binding cavity.On the other hand, the complex between WN191 and Top1 is similar to the previous one with the aromatic moiety totally fitting the DNA groove (Figure 6C).Nevertheless, one can observe that the larger branched arm of WN191 had to switch to another position, putatively decreasing with a small impact on the affinity of this molecule.Moreover, WN198 has a similar structure as WN191 with a copper ion chelating a water molecule.This interaction was difficult to model by molecular mechanics as copper ions are not well parametrized in a classical forcefield.One model was proposed (Figure 6D) that can be easily superimposed on the ligand WN191.Although the binding modes of WN191 and WN198 are almost the same, their activities are slightly different, which may be explained by copper ion playing a role in the stabilization of the ligand before the binding to the DNA groove as discussed in Section 3.

WN198 Induces Autophagy but Not Apoptosis
Apoptosis can be activated after DNA damage [25,26].However, the early and late apoptosis markers, respectively, cleaved caspase 3 and cleaved PARP, were not detected after treatments with WN198 at all concentrations tested, in three adenocarcinoma cell lines, MDA-MB-231, HeLa, HT-29, in contrast to doxorubicin treatment (Figure 7A).γH2AX, an indicator of DNA breaks, was detected after treatment with WN198 and doxorubicin, indicating that WN198 and doxorubicin could induce DNA damage.Untreated cells showed lower γH2AX signals.We then determined whether WN198 could induce autophagy.Several autophagy markers [14] were present after 24h of treatment with 0.5, 1, and 5 µM of WN198 and rapamycin, an inhibitor of the mTOR pathway known to trigger autophagy [27].Beclin-1 was synthesized, and LC3-II (LC3-I in association with phosphatidyl-ethanolamine) was increased while control untreated cells did not show these markers (Figure 7B).

WN198 Induces Autophagy but Not Apoptosis
Apoptosis can be activated after DNA damage [25,26].However, the early and late apoptosis markers, respectively, cleaved caspase 3 and cleaved PARP, were not detected after treatments with WN198 at all concentrations tested, in three adenocarcinoma cell lines, MDA-MB-231, HeLa, HT-29, in contrast to doxorubicin treatment (Figure 7A).γH2AX, an indicator of DNA breaks, was detected after treatment with WN198 and doxorubicin, indicating that WN198 and doxorubicin could induce DNA damage.Untreated cells showed lower γH2AX signals.We then determined whether WN198 could induce autophagy.Several autophagy markers [14] were present after 24h of treatment with 0.5, 1, and 5 µM of WN198 and rapamycin, an inhibitor of the mTOR pathway known to trigger autophagy [27].Beclin-1 was synthesized, and LC3-II (LC3-I in association with phosphatidyl-ethanolamine) was increased while control untreated cells did not show these markers (Figure 7B).

Discussion
Topoisomerases are overexpressed in cancer cells which divide rapidly and have a high frequency of M phase in their cell cycle.This overexpression can be exploited by inhibition of topoisomerases by appropriate drugs that generate a high number of DNA breaks leading to cancer cell death [28][29][30].Cells overexpressing topoisomerases have been shown to better respond to topoisomerase inhibitors [31,32].
Since the discovery of platinum's anticancer properties, platinum-derived drugs have become a mainstay of cancer therapy [2,33].Other metal-based drugs have been designed and developed [3,4], like transition metals from the d-block of the periodic table

Discussion
Topoisomerases are overexpressed in cancer cells which divide rapidly and have a high frequency of M phase in their cell cycle.This overexpression can be exploited by inhibition of topoisomerases by appropriate drugs that generate a high number of DNA breaks leading to cancer cell death [28][29][30].Cells overexpressing topoisomerases have been shown to better respond to topoisomerase inhibitors [31,32].
Since the discovery of platinum's anticancer properties, platinum-derived drugs have become a mainstay of cancer therapy [2,33].Other metal-based drugs have been designed and developed [3,4], like transition metals from the d-block of the periodic table (groups 3 to 12) [1,34,35], as they adopt a wide variety of coordination geometries.Indeed, previous studies have demonstrated organometallic compounds composed of metallic atoms such as copper [9], iron (e.g., ferrocifen/ferroquine [1,36]), ruthenium (e.g., indenoisoquinoline [37] and various complexes [38]), or platinum (e.g., cisplatin [39]), which can be used as efficient anticancer drugs.Among them, copper allows a modification in complexed ligand backbones and better DNA affinity [11,40,41].Some copper derivatives interact with DNA, enhancing DNA damage and antitumor activity [42].Other copper complexes inhibit topoisomerase activity and result in DNA damage, cell cycle arrest, autophagy, and death in cancer cells [13][14][15].Recently, a new class of topoisomerase inhibitors derived from indenoisoquinoline were developed and selected for their capacity to escape efflux transporters implied in cell resistance and for their high stability [16,43].These indenoisoquinoline derivatives are in phase I/II clinical trials [16,43].
In an attempt to develop new efficient organometallic compounds, a copper(II) indenoisoquinoline complex named WN198 was synthesized with a branched triamine moiety attached to the indenoisoquinoline core.In this compound, the triamine ligand copper(II) was inserted at the end of the four-carbon chain as a linker because it was previously shown this moiety enhances the DNA-binding affinity due to many hydrogen bonds [36,44,45].The Cu(II) complex structure displays a mononuclear unit of indenoisoquinolinetriamine and two (ClO 4 ) − .At the center of a distorted square with three nitrogen atoms from the indenoisoquinolinetriamine and one oxygen atom from water are five coordinated Cu(II) ions.
In vitro tests reveal that WN198 inhibits topoisomerase I in a dose-dependent manner starting at 1 µM and with optimum efficiency at 2 µM, while the initial compound (WN191) only starts inhibiting topoisomerase I at 2 µM.Furthermore, WN198 could induce DNA breaks detected by a γH2AX signal.Because H2AX phosphorylation correlates with DNA lesions it is used as a marker.When DNA lesions are triggered, the activation of the DNA damage response (DDR) pathway leads to the phosphorylation of histone H2AX on serine 139 (γH2AX) by phosphoinositide 3-kinase-related kinase family members [46,47].
We could further determine that WN198 interacts with DNA with an apparent DNAbinding constant value higher by a factor of 1.2 compared to the original indenoisoquinoline ligand value, showing that the complexation of the ligand with copper allows a slight improvement in the interaction with DNA.A catalytic mode of inhibition could also occur through the intercalation of WN198 into the DNA as demonstrated by the melting curves and the fluorescence measurements.Indenoisoquinolines are composed of a planar skeleton, and the high affinity of the copper(II) complex for DNA could be the result of the π-cation interaction between the atom of Cu(II) coordinated with ligands and the base pairs [48,49].DNA intercalation of WN198 could impede topoisomerase's access to the DNA fixation sites as seen for other topoisomerase inhibitors, such as anthracyclines [50,51].Due to a strong affinity for DNA duplexes, anthracycline compounds prevent topoisomerase binding to DNA [50,51].Doxorubicin, one of the most effective chemotherapeutic drugs used against solid tumors in the treatment of several cancer types, displays a poison activity at low doses and an intercalating catalytic inhibitory action at high doses.Indenoisoquinoline copper derivative WN197 also efficiently induced MDA-MB-231, HeLa, and HT-29 cell death below 0.5 µM.The planar indenoisoquinoline skeleton of WN197 displays high intercalation into DNA [15].Low doses of WN197 inhibit topoisomerases while, at higher doses, the compound has DNA intercalation properties.However, WN197 exhibits two complexed ligand backbones around a single Cu(II) atom, compared to a single one in WN198.While some of the topoisomerase I inhibitors developed do not use intercalation [52], and knowing DNA-binding and topoisomerase I poisoning activities can be viewed as separate mechanisms [53], our results nevertheless encourage the synthesis of series of indenoisoquinoline drugs composed of a backbone with a four-carbon side chain around a metallic center to keep topoisomerase inhibition and strong DNA interaction properties.
Based on existing structures, we demonstrate by molecular modeling that our drugs interfere with DNA and thus block the activity.Thanks to the structural approach, we can clearly see that the most important moiety is the aromatic intercalating one.
The viability assays showed that low doses between 0.37 and 1.6 µM could induce cell death in breast, cervix, colon, and prostate cancer cell lines, from five highly prevalent adenocarcinomas.The lowest IC 50 values were obtained for cervix and breast triplenegative cancer cell lines at doses below that of the original compound (respectively, by a factor of 1.1 and 3).These IC 50 values are under the values determined for many other topoisomerase I inhibitors [9].Higher doses were necessary to affect the MCF-10A human non-tumorigenic epithelial breast line or Xenopus oocyte G2/M transition and embryonic development.The IC 50 of WN198 on MCF-10A cells was higher compared to WN197, respectively, 9.73 and 1.080 µM, which makes WN198 a better candidate to avoid side effects of chemotherapies on non-cancerous cells.The toxicity characteristics of WN198 need further determination with respect to membrane permeability and the determination of cellular uptake.
Copper complexes or topoisomerase inhibitors arrest the cancer cell cycle in different phases and trigger cell death differently by apoptosis or senescence or have bimodal action through both apoptosis and autophagy [54][55][56].Recently, another copper complex derived from the topoisomerase I inhibitor indenoisoquinoline was shown to arrest the cell cycle in the G2 phase through inhibition of Cdc25C phosphatase necessary to activate MPF (Cdk1/cyclin B) and to trigger cell death by autophagy [9,14,15].WN198 triggers autophagy, shown by the accumulation of Beclin-1 and the formation of LC3-II, but no apoptosis as cleaved markers such as PARP and caspase 3 were not detected in the cell lines MDA-MB-231, HeLa, and HT-29 for doses of WN198 equal to and above the IC 50 .
The influence of indenoisoquinoline amine complexations with other metals should be examined in future studies with the knowledge that low minimum and necessary doses of chemotherapeutic compounds could be useful in circumventing normal cell death and limiting cardiotoxicity, a strategy employed for anthracyclines [57,58].

Human Topoisomerase I In Vitro Activity
Topoisomerase I activity was determined with in vitro drug screening kits (TopoGEN, Inc., Buena Vista, CO, USA assays) based on the relaxation of supercoiled DNA into relaxed DNA as previously described [15].Briefly, the assembled reaction mixture was composed of supercoiled pHOT1 DNA (250 ng), 10× TGS buffer (10 mM Tris-HCl pH 7.9, 1 mM EDTA), human topoisomerase I (5 units), the tested compound, and H 2 O to a final adjusted volume of 20 µL.WN191 and WN198 were tested at concentrations ranging from 0.2 to 2 µM.Camptothecin (10 µM) was used as a positive control (poison inhibitor of topoisomerase I activity), etoposide (100 µM) as negative control (inhibitor of topoisomerase II activity), 1% DMSO alone as vehicle control, and relaxed pHOT1 DNA (100 ng) alone as migration control.All reaction products were submitted to 1% agarose gel electrophoresis with EtBr (0.5 µg/mL) at 100 V for 1 h in TAE buffer (Tris-Acetate-EDTA; pH 8.3).

Ethidium Bromide Competition Test
Titrations of fluorescence were determined as described [36,45].EtBr/WN191 or EtBr/WN198 with a molar ratio of 12.6/10 at concentrations ranging from 0.05 to 10 µM was used in a BPE buffer at pH 7.1.The excitation wavelength was 546 nm and the emission was monitored in the range of 560 to 700 nm (SPEX Fluorolog, Horiba-Jobin Yvon).EtBr displacement IC 50 values were calculated with a fitting function incorporated into GraphPad Prism V6.0.The apparent binding constants were calculated using the equation K app = (1.26(K app (EtBr)/IC 50 ) with K app (EtBr) = 10 7 M −1 .

Molecular Modeling
Receptor structure was the crystal structure of human DNA Top1 in complex with topotecan poison (Figure 4A) (PDB ID: 1K4T, 2.1 Å).All ligands and water molecules were removed except DNA.Simulation of the binding modes of the receptor and the designed compounds was performed using the GOLD docking program [60].GOLD is based on a genetic algorithm and considered the ligand as flexible, while side chains of most residues were kept rigid.For the search procedure, a sphere of 30 Å was centered on the X, Y, and Z coordinates of the topotecan poison (Origin = 22.7 −9.4 26.9).Twenty conformations were generated for each ligand using default parameters.Then, all the different binding poses were scored with the ChemPLP scoring function.Subsequent energy minimization was performed using the AMBER forcefield.Finally, all the molecular representations were sketched using PyMOL [61].

Xenopus laevis Oocyte and Embryo Handling
Xenopus laevis females were obtained from the CRB-University of Rennes I, France.They were housed at PHExMAR, University of Lille.Xenopus were maintained in accordance with the EU Directive 2010/63/EU and the French National Guidelines for Use of Animals for Scientific Purposes.The experimental protocols were approved by the "Comité d'Ethique CEEA-75 en Experimentation Animale Nord-Pas de Calais-France" (Xenopus project number: F59-00913).
Females were anesthetized by immersion in a solution of Tricaine (MS222, Sandoz) at 3 g.L −1 for 1 h.The ovaries were surgically removed and placed in ND96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, pH 7.5) at 19 • C. Stage VI oocytes were harvested by using a 1 h collagenase A treatment (1 mg/mL, Boehringer Mannheim) for 45 min with a manual dissociation under a binocular microscope.Microinjections were performed under a binocular microscope with a Nishiryo positive displacement digital micropipette, in the equatorial region of the oocytes, with 60nl of control DMSO 0.1%, doxorubicin, or WN198 at 0.5, 1, and 5 µM.Meiotic resumption was triggered by the addition of progesterone (4 µM) after microinjection and the addition of a corresponding concentration of doxorubicin or WN198 to the external medium (0.5, 1, and 5 µM).The appearance of a white spot, resulting from the migration of the germinal vesicle at the apex of the pigmented hemisphere of the oocyte, indicated the meiosis process or G2/M transition.Oocytes arrested in prophase I progress to metaphase II and arrest [22,23,62].
For spawning, females were stimulated by a dorsal lymph sac injection of human chorionic gonadotropic hormone (900 U).Mature oocytes were obtained by a slight pressure on the animal's ovaries.Testicles were surgically removed from a male after anesthesia in 5 g.L −1 MS222.In vitro fertilization was performed under gentle agitation in 0.22 µm filtered water with doxorubicin or WN198 at 0.5, 1, and 5 µM or not.Eggs were deganguated with 2% L-cysteine with doxorubicin or WN198 or not for 10 min and rinsed three times.Embryos were kept at 23 • C in daily renewed solution with doxorubicin or WN198 or not.Embryo stages were identified using the Nieuwkoop and Faber table (Nieuwkoop and Faber, 1994) [24]: morula (segmentation, stage 6.5, 3 h 30 min after fertilization), gastrula (stage 10, 9 h after fertilization), neurula (stage 18, 19 h 45 min after fertilization), and tailbud (organogenesis, stage 22, 24 h after fertilization).

Conclusions
A new copper(II) complex containing an indenoisoquinoline scaffold was synthesized.The molecular structure of WN198 was confirmed by single-crystal X-ray diffraction analysis.WN198 displays a strong DNA interaction and kept a topoisomerase I inhibitory activity as detected by in vitro tests.
The compound exerts excellent cytotoxic activities against five adenocarcinoma cancer cell lines at a lower concentration compared to other classical topoisomerase drugs used in chemotherapies.It is particularly efficient against MDA-MB-231 (triple-negative breast cancer) cell line proliferation with an IC 50 of 0.37 µM.The IC 50 on non-cancerous cell line MCF-10A is significantly high compared to other copper complexes as topoisomerase inhibitors and no toxicity was detected below 5 µM for Xenopus oocyte maturation and embryo development.
WN198 appears to be a new efficient drug to counteract cancer cells at low doses.WN198 could benefit patients overexpressing topoisomerases, sensitize cancer cells to

Figure 2 .
Figure 2. Synthesis of the ligand WN191 and the Cu(II) complex WN198.The structure of WN198 was established by single-crystal X-ray analysis.The drawing is displayed in Figure3.The Cu complex crystallizes in the triclinic space group P1̅ with two formula units per unit cell.The XRD results show that the Cu(II) complex consists of a mononuclear [Cu(II)L(H2O)(CH3OH)] 2− (where L is the indenoisoquinolinetriamine ligand) unit and two (ClO4) − .The five coordinated Cu(II) ions occupy the center of the distorted square base of the pyramid consisting of three nitrogen atoms from the indenoisoquinolinetriamine ligand and one oxygen atom of a water molecule (1.995 Å).One O atom of (CH3)OH completes the pyramidal environment with an axial bond distance of Cu-O = 2.333 (7) Å.

Figure 2 . 18 Figure 2 .
Figure 2. Synthesis of the ligand WN191 and the Cu(II) complex WN198.The structure of WN198 was established by single-crystal X-ray analysis.The drawing is displayed in Figure 3.The Cu complex crystallizes in the triclinic space group P1 with two formula units per unit cell.The XRD results show that the Cu(II) complex consists of a mononuclear [Cu(II)L(H 2 O)(CH 3 OH)] 2− (where L is the indenoisoquinolinetriamine ligand) unit and two (ClO 4 ) − .The five coordinated Cu(II) ions occupy the center of the distorted square base of the pyramid consisting of three nitrogen atoms from the indenoisoquinolinetriamine ligand and one oxygen atom of a water molecule (1.995 Å).One O atom of (CH 3 )OH completes the pyramidal environment with an axial bond distance of Cu-O = 2.333 (7) Å.

Figure 3 . 18 Figure 2 .
Figure 3. Molecular structure of Cu complex WN198 showing the local geometry around the copper and ligand.Selected bond lengths (

Figure 4 .
Figure 4. Non-cancerous cell viability under WN198 treatment.(A) MCF-10A IC50 values are expressed as the mean ± SD of three independent experiments.Cisplatin and WN197 were used as control.(B) Xenopus oocyte G2/M transition was scored by the determination of a white spot at the animal pole showing the progression from prophase I to metaphase II (meiosis maturation) 12 h after microinjection and balneation with corresponding drugs (0.5, 1, 5 µM) and external stimulation by progesterone as a natural inducer (4 µM).Experiments were performed on 10 to 20 oocytes from three different females.(C)The viability of Xenopus embryos was followed after incubation with corresponding drugs (0.5, 1, 5 µM).Stages were identified using the Nieuwkoop and Faber table[24]: segmentation, (3 h 30 min after fertilization), gastrulation (9 h after fertilization), neurulation (19 h 45 min after fertilization), tailbud (24 h after fertilization).Experiments were performed on two independent fertilizations and 10 to 40 embryos in each condition.

Figure 4 .
Figure 4. Non-cancerous cell viability under WN198 treatment.(A) MCF-10A IC 50 values are expressed as the mean ± SD of three independent experiments.Cisplatin and WN197 were used as control.(B) Xenopus oocyte G2/M transition was scored by the determination of a white spot at the animal pole showing the progression from prophase I to metaphase II (meiosis maturation) 12 h after microinjection and balneation with corresponding drugs (0.5, 1, 5 µM) and external stimulation by progesterone as a natural inducer (4 µM).Experiments were performed on 10 to 20 oocytes from three different females.(C) The viability of Xenopus embryos was followed after incubation with corresponding drugs (0.5, 1, 5 µM).Stages were identified using the Nieuwkoop and Faber table[24]: segmentation, (3 h 30 min after fertilization), gastrulation (9 h after fertilization), neurulation (19 h 45 min after fertilization), tailbud (24 h after fertilization).Experiments were performed on two independent fertilizations and 10 to 40 embryos in each condition.

Figure 5 .
Figure 5. WN191 and WN198 inhibited human topoisomerase I activity in a dose-dependent manner.Topoisomerase I (Top1) activity is determined by in vitro assays after the addition of either (A) WN198 or (B) WN191 at different concentrations (0.2, 0.5, 1, and 2 µM, lanes 5-8).Relaxed DNA ( R DNA, lane 1) or supercoiled DNA ( SC DNA, lane 2) is used as migration control.The Top1 activity control allowing the relaxation of SC DNA is in lane 3. DMSO is the solvent control (5%, lane 4).SC DNA is used in all other reactions in the presence of Top1.(C) Etoposide (VP-16, 50 µM; topoisomerase II (Top2) poison, lane 1) is the negative control of Top1 activity inhibition, and camptothecin (CPT, 10 µM; Top1 poison, lane 2) the positive control of Top1 activity inhibition.After topoisomerase reactions, DNA is run in a 1% agarose gel, stained with ethidium bromide (0.5 µg/mL), and visualized under UV light.

Figure 6 .
Figure 6.Interaction models of the studied ligands and Top1.(A) Crystal structure of the topotecan poison (grey sticks) fitting the DNA groove (orange sticks) of Top1 (brown helices) (PDB ID: 1K4T, 2.1 Å). (B) The binding mode of WN170 (salmon sticks) is similar to topotecan's binding mode.The main interaction is taking place inside the DNA groove.(C) The interaction model of WN191 (yellow sticks) is slightly different from WN170 as the larger branched arm has to switch down.The main part of the infarction is occurring through the aromatic ring.(D) The binding mode of WN198 (pink sticks with the copper molecule as a blue ball) is similar to the binding mode of WN191.

Figure 6 .
Figure 6.Interaction models of the studied ligands and Top1.(A) Crystal structure of the topotecan poison (grey sticks) fitting the DNA groove (orange sticks) of Top1 (brown helices) (PDB ID: 1K4T, 2.1 Å). (B) The binding mode of WN170 (salmon sticks) is similar to topotecan's binding mode.The main interaction is taking place inside the DNA groove.(C) The interaction model of WN191 (yellow sticks) is slightly different from WN170 as the larger branched arm has to switch down.The main part of the infarction is occurring through the aromatic ring.(D) The binding mode of WN198 (pink sticks with the copper molecule as a blue ball) is similar to the binding mode of WN191.

Table 2 .
Melting curves and fluorescence measurements for WN191 and WN198.
Apparent binding constants were measured by fluorescence using [EtBr]/[DNA] = 1.26.Data are the mean of at least three independent experiments.

Table 2 .
Melting curves and fluorescence measurements for WN191 and WN198.
ClO 4 ) 2 .6H 2 O (273 mg, 0.74 mmol) in MeOH (7 mL).After stirring at room temperature for 24 h, the mixture was filtered and washed with methanol.The powder was recrystallized in a mixture of methanol/ether to give orange crystals (367 mg, 70%).

Table 3 .
Crystal data and structure refinement for Cu complex WN198.